Increased exposure to antibiotics has led to the dissemination of genes conferring resistance to antimicrobial metabolites throughout human microbiomes globally via horizontal gene transfer (HGT). This has resulted in the emergence of new resistant strains leading to a rising epidemic of deaths from previously treatable infections. Evidence suggests that before the age of anthropogenic antibiotic use, microbes living within a community produced antibiotic metabolites and, subsequently, maintained such genes for several useful functions and a balance of diversity in nature. The question of the origin of these resistant genes is difficult to answer, but with continued advancements in ancient genomic analysis, researchers have developed methods of acquiring a more accurate representation of the microbiome associated with our human ancestors by extracting fossilized microbial specimens from dental calculus and directly sequencing the metagenomes. This thesis outlines the production of taxonomic and functional profiles of 20 different human and non-human oral microbiome samples using metagenomics tools originally developed for living individuals, altered for use with ancient microbial specimens. Putative antimicrobial resistant (AMR) genes derived from these profiles were reconstructed and conserved functional regions were identified. From the data that is available regarding the human microbiome from a range of time points throughout history dating back to Neanderthal specimens, it is possible to elucidate relationships between these AMR genes and to better understand the evolutionary trajectory of antibiotic resistance.

Parmi tous les animaux domestiques, le cheval est sans aucun doute celui ayant le plus influencé l’histoire des peuplements humains. Le cheval domestique a d’abord fourni à de nombreuses civilisations des ressources primaires essentielles telles que la viande et le lait. Utilisé pour sa force physique et comme moyen de transport, il a eu de profondes conséquences sur les mouvements de personnes et de biens ainsi que sur la diffusion de cultures et d’idées à travers l’Eurasie. Le cheval a ainsi fortement contribué à l’expansion de sociétés et d’empires pendant des millénaires, et ce jusqu’au vingtième siècle. Les différentes étapes de la domestication du cheval restent cependant mal comprises d’un point de vue archéologique et sont complexes à retracer à partir des données génétiques recueillies sur les races chevalines actuelles. L’émergence de la génomique ancienne au début des années 2010 a révolutionné la biologie de l'évolution, en donnant un accès direct à l’histoire des populations anciennes et actuelles. Elle est donc particulièrement adaptée pour étudier la transition historique induite par la domestication du cheval. En s'appuyant sur les dernières avancées en matière d’extraction d'ADN ancien et des technologies de séquençage d’ADN à haut débit, ce travail de doctorat vise à décrypter les modifications génétiques sous-jacentes au processus de domestication du cheval. Pour se faire, nous avons généré le plus grand jeu de données génomiques anciennes jamais rassemblées sur un organisme non humain. Celles-ci ont révélé que les chevaux domestiqués pour la première fois à Botai, dans le nord du Kazakhstan, il y a environ 5 500 ans, ne sont pas les ancêtres des chevaux domestiques ayant vécu pendant ces dernières ~4 100 années. Ce sont les ancêtres des chevaux de Przewalski, que l’on pensait jusqu’alors totalement sauvages. Cette découverte inattendue suggère qu'un remplacement majeur de la population de chevaux domestiques a eu lieu au cours du troisième millénaire avant notre ère, contribuant probablement à faire entrer l'humanité dans l'âge du Bronze. En outre, ces trois années de recherche ont permis d'identifier les signatures génétiques associées à différentes stratégies d’élevage du cheval et ont révélé les dynamiques évolutives en jeu lors des étapes clés de la domestication. En particulier, il ressort des analyses de génomes anciens que les chevaux ibériques n’ont contribué que marginalement à la création du cheval domestique tel qu’on le connaît aujourd'hui. Ce travail de thèse a par ailleurs permis de détecter une influence croissante des chevaux perses dès le début du Moyen Age. / Among all domesticates, the horse can confidently be considered as the animal that most impacted the history of human dynamics. Once they domesticated the horse, human civilizations got hold of essential domestication products including meat and milk, but also invaluable secondary products, such as fast transportation and powerful workforce. The horse thus deeply enhanced the circulation of people, goods, culture and ideas, promoting the spread of vast military and political units across Eurasia up until the 1900s. The various steps underpinning horse domestication are however difficult to track in the archaeological record and still poorly understood based on patterns of DNA variation among modern breeds. In the last decade, the advent of ancient genomics has revolutionized evolutionary biology by providing a direct window into the past history of populations. Ancient genomics therefore provides the necessary time travel machine to investigate the key historical transition in the history of humankind that was induced by the horse domestication. Leveraging the latest advances in ancient DNA recovery and High-Throughput sequencing technologies, this PhD project aimed at deciphering the genetic changes underlying the horse domestication process by generating the largest ancient genome dataset for a non-human organism, spanning the whole temporal and geographic range of horse domestication. This dataset revealed that horses first herded at Botai in Northern Kazakhstan ~5,500 years ago are not the ancestors of modern domestic horses but instead of modern Przewalski’s horses, previously thought to represent last true wild population on Earth. This major discovery also suggests that a swift genomic replacement in the domestic stock took place in the third millennium BCE, probably contributing to precipitating humankind into a new metal era, the Bronze Age. Additionally, this PhD work identified the genetic signatures associated with different management strategies and the evolutionary dynamics at play within distinct domestication stages. In particular, we were able to rule out Iberia as a major contributor to the modern domestic stock and moving towards more recent times, we characterized the growing influence of Persian-like horses starting in the early Middle Ages.

Bio-archaeology is the study of biological remains found at sites of archaeological interest. It is an interdisciplinary science employing different scientific fields including physical anthropology, geography, archaeology and genetics. Genetic analysis includes ancient DNA (aDNA) studies, now a specialised field in genetics. This approach was used to analyse human skeletal material of eight individuals from various Iron Age archaeological sites in southern Africa. Included in this sample is a naturally mummified individual from Tuli, in Botswana. The context of the specimens found in the Limpopo Province (Thulamela), as well as their cultural links with the Zimbabwe Culture Complex (which includes Mapungubwe and Khami) suggests that some gene exchange might have occurred. While this is not the first aDNA study on southern African samples, it is the first aDNA study based on southern African Iron Age human individuals and also included a naturally mummified individual. Morphometric and morphological analyses have indicated the age at death, sex and health status of the individuals, and the context in which they were found has helped in assessing their cultural affinity. Bone samples were analysed in a specialized aDNA laboratory at the Centre for Evolutionary Medicine in Switzerland. Following DNA extraction, ancestry-specific mitochondrial DNA was amplified from all samples and was compared to that of modern sub-Saharan Africans whose data were accessed from GenBank. Some individuals show (maternal) genetic similarities to present-day Sotho/Tswana groups. The male individual from Thulamela aligns somewhat more with the groups from the west and the female with the eastern peoples. Two Early Iron Age individuals from Happy Rest presented some similarities to the Khoesan peoples. Genetic-sex determination efforts were inconclusive for all individuals. The purpose of this study was to place the Thulamela individuals within the context of the genetic diversity in South Africa. It was noted that the introduction of genetic material from the early Sotho/Tswana was gradual in the case of Thulamela. Two other individuals from Happy Rest, who were contemporaries of each other, showed very little genetic variation and it can be said that their maternal DNA was of the same (possibly Khoesan) origin. Further resolution in haplotype assignment will be done in future. These temporally and spatially dispersed individuals can only provide a glimpse into the population interactions of the Iron Age that may have partially shaped the immense genetic diversity of present-day southern Africa. / Dissertation (MSc)--University of Pretoria, 2014. / tm2015 / Anatomy / MSc / Unrestricted

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material Over course of the last decade, the field of ancient DNA has been transformed by the advent of highthroughput sequencing. In specimens with exceptional DNA preservation, this allowed whole nuclear genomes of extinct organisms to be sequenced and analyzed. To deal with material with suboptimal DNA preservation and high levels of external modern DNA contamination as is common in intensively handled museum specimens, significant challenges remain. In this thesis I developed laboratory and in silico methods to make highly contaminated biological material amenable to genetic research and explore the structure and decay mechanisms of ancient DNA. Chapter 1 illustrates the development of a library preparation method for high-throughput sequencing that selects for DNA molecules containing uracils, a damaged base typically found in ancent DNA, in order to enrich authentic ancient molecules against a background of modern contamination. The method was applied to Neanderthal samples from Gibraltar demonstrating its suitability for recovering DNA sequences from material that shows very poor DNA preservation and that is particularly strongly contaminated with modern human DNA. Chapter 2 describes a new method to reconstruct the native double-stranded DNA molecules and overhang structures in DNA isolated from a Neanderthal specimen by combining deep sequencing of low-input single-stranded DNA libraries with in silico sequence matching. I analyzed patterns of nucleotide substitutions and base composition separately in the different structures found and showed also that the method can be applied to modern sources of fragmented DNA, specifically to cell-free DNA isolated from present-day blood samples.

Forensic scientists and ancient DNA researchers face similar challenges with respect to genetic information acquisition and analysis. However, these communities differ in one critical aspect: while forensic science is regulated by the strict guidelines of the judicial community, ancient DNA is a research-based academic field free to explore emerging technologies as they arise. This thesis investigates the application of two methodologies, developed in ancient DNA research, to challenging extracts, in hopes of modernizing forensic models while maintaining compatibility with current standards. The first chapter focuses on blunt-end sequencing library preparation protocols previously optimized for ancient DNA specimens. Forensically-relevant extracts were converted into libraries and typed by short tandem repeats (STR) amplification. When compared to STR profiles from pre-library extracts, a significant decrease in the quality was observed, in the form of allelic drop-out, heterozygous peak imbalance and increased stutter ratios. The second chapter discusses the efficacy of two enzymatic DNA repair methods, “PreCR® Repair” and “Nelson”, on typical ancient DNA specimens. Based on endogenous sample content, fragment length variation and base misincorporation rates, some DNA repair was reported when using PreCR®. However, the use of the Nelson protocol is not recommended for use in its current state. Both sequencing library preparation and enzymatic DNA repair show potential application to forensic evidential material, but require further analyses to confirm hypotheses and observations outlined in this thesis. / Thesis / Master of Science (MSc)

The analysis of kinship from ancient DNA (aDNA) data has the potential to provide insight into social structures of prehistoric societies. Kinship analysis is gaining popularity as optimised wet-lab methods allow for studies with sample sizes on the level of whole cemeteries. However, the specifics of ancient DNA require different methods than what would be used for modern DNA. A common way is to use the sites that are identical-bydescent (IBD), however, detecting these is often a challenging task since it is not easy to determine whether a shared locus between two individuals is inherited from the ancestor or if another factor caused the similarity. Most methods used in the field are able to identify up to 2nd or 3rd degree relatives from aDNA data but do not distinguish between different types of relationship for the same degree, for instance not being able to differentiate between parentoffspring and full sibling-sibling relationship in first degree. The aDNA kinship methods often use either of window-based or single-site approaches, however, these two approaches have not been compared formally before in terms of effectivity and efficiency. In this work, READv2 is presented as a re-implementation of a popular kinship analysis method for aDNA studies with additional features such as accepting .bed files as input, which take up less space than the previous input type, plain-text .tped files. It is shown that the new version works more efficiently in terms of runtime. However, the memory requirements seem to be increased with the new implementation. Furthermore, a window-based approach is compared with the single-site approach of READv2, as well as varying window sizes, with benchmarked simulation data which contains approximately 700 individuals with known 1st degree, 2nd degree and 3rd degree relationships. According to the comparison, the sensitivity of the method does not vary between the approaches and different window sizes for high coverages. However, the single-site approach has been shown to be the superior one by a small margin for lower coverages. In addition to these, using the variance of non-shared alleles in windows along the genome has been used to implement a method to differentiate different first-degree relationships, parent-offspring and siblings. The method is tested with an independent dataset from the 1000 Genomes Project which shows that the proposed method is able to work with different datasets with varying sets of SNPs. Nevertheless, the first-degree classification method requires further analyses to determine the stress-point where the True Positive rates for both categories start to drop. Additionally, some necessary changes and decisions are required for READv2 to be a user-friendly method that can be used by other researchers. The preliminary release of READv2, including example data as well as instructions to install the necessary packages and to run the algorithm can be found in https://github.com/GuntherLab/READv2/releases/tag/READ.

No description available.

Pandemics of plague have reemerged multiple times throughout human history with tremendous mortality and extensive geographic spread. The First Pandemic (6th - 8th century) devastated the Mediterranean world, the Second Pandemic (14th - 19th century) swept across much of Afro-Eurasia, and the Third Pandemic (19th - 20th century) reached every continent except for Antarctica, and continues to persist in various endemic foci around the world. Despite centuries of historical research, the epidemiology of these pandemics remains enigmatic. However, recent technological advancements have yielded a novel form of evidence: ancient DNA of the plague bacterium Yersinia pestis. In this thesis, I explore how genomic data can be used to unravel the mysteries of when and where this disease appeared in the past. In particular, I focus on phylogenetic approaches to studying this 'small microbe' with 'big data' (i.e. 100s - 1000s of genomes). I begin by describing novel software I developed that supports the acquisition and curation of large amounts of DNA sequences in public databases. I then use this tool to create an updated global phylogeny of Y. pestis, which includes ~600 genomes with standardized metadata. I devise and validate a new approach for temporal modeling (i.e. molecular clock) that produces robust divergence dates in pandemic lineages of Y. pestis. In addition, I critically examine the questions that genomic evidence can and cannot address in isolation, such as whether the timing and spread of short-term epidemics can be confidently reconstructed. Finally, I apply this theoretical and methodological insight to a case study in which I reconstruct the appearance, persistence, and disappearance of plague in Denmark during the Second Pandemic. The three papers enclosed in this sandwich-thesis contribute to a larger body of work on the anthropology of plague, which seeks to understand how disease exposure and experience change over time and between human populations. Furthermore, this dissertation more broadly impacts both prospective studies of infectious disease, such as environmental surveillance and outbreak monitoring, and retrospective studies, which seek to date the emergence and spread of past pandemics. / Dissertation / Doctor of Philosophy (PhD) / The Plague is a disease that has profoundly impacted human history and is responsible for some of the most fatal pandemics ever recorded. It may surprise many to know that this disease is not a bygone of a past era, but in fact is still present in many regions of the world. Although researchers have been studying plague for hundreds of years, there are many aspects of its epidemiology that are enigmatic. In this thesis, I focus on how DNA from the plague bacterium can be used to estimate where and when this disease appeared in the past. To do so, I reconstruct the evolutionary relationships between modern and ancient strains of plague, using publicly available data and new DNA sequences retrieved from the skeletal remains of plague victims in Denmark. This work offers a new methodological framework for large-scale genetic analysis, provides a critique on what questions DNA evidence can and cannot answer, and expands our knowledge of the global diversity of plague.

<p>Ancient DNA has the power to elucidate ecological and evolutionary relationships that were previously only quantifiable by proxy. This work details both a metagenetic and a targeted metagenomic study of ancient sedimentary DNA. By using DNA to investigate the plants and animals present in twelve different time points, we describe the nature of the ecological change over the Pleistocene-Holocene transition. We show that as the stability of the habitat degraded due to climate change, the dominant plant communities exhibited a shift from functional groups such as forbs to shrubs and trees. As this cascading change consequently affected the animal communities, we demonstrate the decline, extinction, and replacement of a variety of megafaunal species and mammoths. As well, we provide a proof-ofconcept for the targeted oligonucleotide enrichment for ancient sedimentary DNA. By processing the same DNA extracts with targeted enrichment, we show that metagenomic soil DNA can provide the same taxonomic fingerprint unique to each time period even using different genetic loci. This unique pattern can be used as a reference in future studies. Although the oligonucleotide baits did not yield the composition of taxa that we expected, the oligonucleotide baits did increase the eukaryotic fraction of DNA extracts by up to 50%. Overall, this technique is open to further study and has fantastic potential to redefine the metagenomic work of ancient DNA soil cores.</p> / Master of Science (MSc)

Yes / We examined six skeletons from mediaeval contexts from two sites in England for the presence of Mycobacterium leprae DNA, each of the skeletons displaying osteological indicators of leprosy. Polymerase chain reactions directed at the species-specific RLEP multicopy sequence produced positive results with three skeletons, these being among those with the clearest osteological signs of leprosy. Following in-solution hybridization capture, sufficient sequence reads were obtained to cover >70% of the M. leprae genomes from these three skeletons, with a mean read depth of 4–10×. Two skeletons from a mediaeval hospital in Chichester, UK, dating to the 14th–17th centuries AD, contained M. leprae strains of subtype 3I, which has previously been reported in mediaeval England. The third skeleton, from a churchyard cemetery at Raunds Furnells, UK, dating to the 10th to mid-12th centuries AD, carried subtype 3K, which has been recorded at 7th–13th century AD sites in Turkey, Hungary and Denmark, but not previously in Britain. We suggest that travellers to the Holy Land might have been responsible for the transmission of subtype 3K from southeast Europe to Britain. / Funded by a studentship awarded by Majlis Amanah Rakyat (MARA) to A.K. and by the University of Bradford and the University of Manchester.